cDNA sequence transcribing an mRNA encoding the terminal oxidase associated with carotenoid biosynthesis, and uses thereof

ABSTRACT

The invention concerns a cDNA (complementary deoxyribonucleic acid) sequence represented by SEQ ID NO: 1, transcribing a mRNA (messenger deoxyribonucleic acid), itself coding for the TOCB (terminal oxydase associated with carotenoid biosynthesis) represented by SEQ ID NO: 2, and the complementary sequence of SEQ ID NO: 1, vectors transforming cell, plant or fragment of plant, and the method for modifying the production of carotenoids in a plant.

The invention relates to a DNA (deoxyribonucleic acid) sequencedescribed by SEQ ID NO:1, transcribing an mRNA (messengerdeoxyribonucleic acid), itself encoding the TOCB (Terminal Oxidaseassociated with Carotenoid Biosynthesis) enzyme described by SEQ IDNO:2, and to vectors for transforming a cell, plant or fragment of aplant, and a process for modifying the production of carotenoids in aplant.

Carotenoids are lipophilic pigments synthesized in plants, fungi andbacteria. In photosynthetic tissues, carotenoids serve as an additionallight-absorbing pigment and especially provide photoprotection againstfree radicals, such as singlet oxygen.

In plants and certain microorganisms, the carotenoid biosynthesis routeproduces carotenes, xanthophylls and derivatives thereof. Thesecompounds are synthesized from phytoene which is modified by successivedehydrogenation reactions to give phytofluene, zeta-carotene,neurosporene and then lycopene. Lycopene accumulates in certain cases,for example giving the red pigment of tomatoes, or is more generallyfound in a form modified by cyclization, to form alpha- orbeta-carotene. These cyclized carotenoids are the precursors of vitaminA, and may accumulate or give xanthophylls by oxidation reactions, thesexanthophylls being yellow, pink, orange or red pigments.

The successive steps of dehydrogenation of phytoene are catalyzed inmost microorganisms by a single enzyme known as phytoene desaturaseCRTI. In plants and cyanobacteria, two related enzymes exist. The first,known as phytoene desaturase (PDS), catalyzes the conversion of phytoeneto phytofluene and then into zeta-carotene. The second, known aszeta-carotene desaturase (ZDS), catalyzes the conversion ofzeta-carotene into neurosporene and then into lycopene. Each of thesedehydrogenation reactions requires the transfer of two electrons and twoprotons from the substrate to an acceptor. These dehydrogenationreactions thus require enzymes, known as structural enzymes, andco-factors, which are intermediates in the redox reactions.

The inventors of the present invention have discovered a new geneencoding an enzyme known as TOCB (Terminal Oxidase associated withCarotenoid Biosynthesis), which is involved in carotenoid biosynthesis.It appears that this enzyme is placed in the membranes of chloroplastsand is essential for the correct functioning of PDS.

A first subject according to the invention thus relates to a DNAsequence comprising at least one coding region consisting of:

-   -   the nucleotide sequence represented by SEQ ID NO:1 transcribing        an mRNA, this mRNA encoding the TOCB (Terminal Oxidase        associated with Carotenoid Biosynthesis) enzyme described by SEQ        ID NO:2,    -   the modified nucleotide sequence of the sequence SEQ ID NO:1, as        described above, particularly by mutation and/or addition and/or        deletion and/or substitution of one or more nucleotide(s), this        modified sequence transcribing an mRNA which itself encodes the        TOCB described by SEQ ID NO:2, or encoding a modified protein of        said TOCB, said modified protein having enzymatic activity which        is equivalent to that of the TOCB represented by SEQ ID NO:2.

In particular, the invention relates to the coding sequences of tomatoTOCB, identified by SEQ ID NO:5, and of capsicum TOCB, identified by SEQID NO:3, respectively, and any derived sequence obtained by modifyingthese sequences.

The gene encoding TOCB is a duplex DNA, comprising introns and exons.The sequence SEQ ID NO:1 is the complementary strand (without theintrons) or cDNA, corresponding to the DNA strand transcribing the mRNAencoding TOCB.

The expression “equivalent enzymatic activity” means that, although someof the portions of the enzyme may be structurally modified, it isnevertheless capable of modifying its substrate. Its activity issubstantially the same as that of the native enzyme. It will beunderstood that this enzyme cannot be modified at its active site.Consequently, any modification made to the native sequence, by addition,deletion or substitution of one or more amino acids, is understood asgiving rise to an equivalent enzymatic activity insofar as the activityof the native protein is not affected by these modifications.

A second subject according to the invention relates to a DNA sequencecomprising at least one coding region consisting of:

-   -   the complementary nucleotide sequence represented by SEQ ID        NO:1, this sequence transcribing an antisense mRNA capable of        pairing with the mRNA transcribed by the complementary sequence        of SEQ ID NO:1,    -   the modified nucleotide sequence of the sequence described        above, by mutation and/or addition and/or deletion and/or        substitution of one or more nucleotide(s), this modified        sequence transcribing an antisense mRNA capable of pairing with        an mRNA mentioned above,    -   a fragment of one of the nucleotide sequences mentioned above,        said fragment transcribing an antisense mRNA capable of pairing        with the mRNA encoded by the complementary sequence of SEQ ID        NO:1.

The term “DNA” may be understood as meaning complementary DNA (or cDNA),i.e. the copy of the mRNA in its DNA form by virtue of the action of areverse transcriptase. The cDNA does not comprise the introns of the DNAsequences.

In the present invention, the expression “capable of pairing” means thefact that, under given hybridization conditions, the complementarynucleotide sequences pair up. A person skilled in the art clearly knows,depending on the hybridization conditions used, what percentage ofidentity the sequences must have in order for a pairing or ahybridization to be able to take place. The stringency conditions forobtaining a pairing of similar sequences are, for example, ahybridization in 50% formamide at 35° C. As regards the hybridizationconditions, reference will be made in particular to the article“Molecular Cloning, a laboratory manual, second edition, Sambrook,Fritch & Maniatis, 1989, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., USA”.

In the present invention, the expression “modified nucleotide sequence”means any nucleotide sequence which has a degree of identity with thereference sequence of less than 100%.

According to one preferred embodiment according to the invention, themodified nucleotide sequences according to the present inventioncomprise approximately at least 70% and better still at least 80% ofnucleotides that are identical to those of the nucleotide sequencerepresented by SEQ ID NO:1, or of its complementary sequence.

The expression “nucleotide identity” means the comparison, when the twostrands are aligned, of the sequence of identical nucleotides present onthe two strands. Consequently, by reducing to the total number ofnucleotides, the percentage of identical nucleotides, i.e. thenucleotide identity, is obtained.

A third subject according to the invention relates to an mRNAtranscribed from the DNA sequence according to the definition of thefirst subject, and more particularly transcribed from the DNA sequencerepresented by SEQ ID NO:1, said mRNA encoding the TOCB enzyme describedby SEQ ID NO:2, or a fragment or a modified protein of the enzyme, andhaving activity which is equivalent to that of said enzyme in the plant.

A fourth subject according to the invention relates to an antisense mRNAtranscribed from the DNA sequence according to the second subject of theinvention, comprising nucleotides which are complementary to all or aportion of the nucleotides constituting the native mRNA, and which arecapable of pairing with said mRNA.

The expression “antisense mRNA” means an RNA sequence which iscomplementary to a base sequence of a corresponding mRNA, which iscomplementary in the sense that each base (or the majority of the bases)in the antisense sequence (reading in the 3′ to 5′ direction) is capableof pairing with the corresponding base (G with C, A with U), in the mRNAsequence reading in the 5′ to 3′ direction.

A fifth subject according to the invention relates to a protein with theactivity of the TOCB enzyme described by SEQ ID NO:2, or any modifiedprotein of said TOCB enzyme, particularly by addition and/or deletionand/or substitution of one or more amino acids, or any fragment derivedfrom the TOCB enzyme or from a modified sequence of the enzyme, saidfragment or modified sequence having enzymatic activity which isequivalent to that of the TOCB enzyme.

A sixth subject according to the invention relates to a complex formsbetween an antisense mRNA defined in the fourth subject according to theinvention, and an mRNA encoding a TOCB enzyme in the plant.

A seventh subject according to the invention is a recombinant DNAcomprising a DNA sequence defined in the first subject according to theinvention, said sequence being inserted into a heterologous sequence,said sequences transcribing all or a portion of an mRNA sequenceencoding all or a portion of the TOCB enzyme, this enzyme havingenzymatic activity which is equivalent to that of the TOCB enzyme of theplant.

According to the present invention, the expression “heterologoussequence” means any sequence which may be cut by enzymes, and whichconsequently serves to insert other sequences with diverse activities.

An eighth subject according to the invention is a recombinant DNAcomprising a DNA sequence defined in the second subject according to theinvention, said sequence being inserted into a heterologous sequence,said sequences transcribing all or a portion of an antisense mRNAsequence capable of pairing with an mRNA encoding a TOCB enzyme in theplant.

A ninth subject according to the invention is a recombinant DNA definedin the seventh or eighth subject according to the invention, comprisingthe elements required to control the expression of the insertedsequence, in particular a promoter sequence and a sequence for stoppingthe transcription of said sequences.

A tenth subject according to the invention relates to a vector fortransforming plants, which is adapted to increase carotenoidbiosynthesis, comprising all or a portion of the nucleotide sequence ofSEQ ID NO:1 as defined in the first subject according to the invention,encoding all or a portion of an enzyme involved in carotenoid synthesis,represented by SEQ ID NO:2, preceded by an origin of replication of thetranscription of the plants, such that the vector can generate mRNA inthe plant cells.

An eleventh subject according to the invention relates to a vector fortransforming plants, which is adapted to reduce or stop carotenoidbiosynthesis, comprising all or a portion of the strand of thenucleotide sequence which is complementary to SEQ ID NO:1 as defined inthe second subject according to the invention, preceded by an origin ofreplication of the transcription of the plants, such that thecomplementary strand transcribed can pair with the mRNA encoding theplant's TOCB enzyme involved in carotenoid synthesis.

The invention may thus be used to modify carotenoid synthesis, forexample to increase or reduce, or even stop, the production of thecolors associated with the dehydrogenation of phytoene. For example, theinhibition of the red color in fruit such as tomatoes, by transformationwith a vector comprising an antisense sequence, gives a fruit with anattractive color close to yellow, for instance that of certaincapsicums. Yellow tomatoes of this kind already exist, but the presentinvention provides a means for transferring the characteristic colorinto lines, without a prolonged reproduction program being necessary andas a result possibly giving rise to an impairment of othercharacteristics of the plant.

The increase in carotenoid synthesis by transformation with a vectorcomprising a sense sequence may make it possible to produce tomatoes ofa more intense red color, which consumers may find more appetizing. Theinvention may also serve to introduce a red color into a plant, otherthan into the fruit. The increase in carotenoid synthesis in a plant maybe carried out by inserting one or more functional copies of thecomplementary DNA gene, or the whole gene, under the control of afunctional promoter into the plant cells.

The vectors for transforming plants to reduce or stop carotenoidsynthesis, i.e. the antisense vectors, may be very short. In onepreferred embodiment, homologous base sequences having a length of atleast 10 bases will be selected. There is no theoretical upper limit tothe base sequence; it may be as long as the mRNA produced by the plant.However, in one very preferential embodiment, sequences between 100 and1000 bases long will be used.

It is known that the mutant plants in which the TOCB gene is inactivehave a variegated appearance; the plants are green and white. Anapplication of the antisense strategy is proposed, which is directedtoward eliminating the production of mRNA and thus of the TOCB protein,which would be directed toward producing plants with variegated foliagesuch as, for example, ornamental plants, for instance Nicotiana orPetunia or any other ornamental plant, which lends itself to genetictransformation and which could receive an antisense construct for thepurpose of preventing the production of the TOCB protein.

The DNA recombination products may be manufactured using standardtechniques. For example, the DNA sequence to be transcribed may beobtained by treating a vector containing said sequence with restrictionenzymes to cut out the appropriate segment. The transcription DNAsequence may also be generated by cyclizing and binding syntheticoligonucleotides or by using synthetic oligonucleotides in a PCR(“polymerase chain reaction”) to generate restriction sites at each end.The DNA sequence is then cloned into a vector containing a startpromoter sequence and a stop sequence. If it is desired to obtain anantisense DNA sequence, the cloning will be carried out so that the DNAsequence cut out is inverted relative to its orientation in the strandfrom which it was cut out.

In a recombination product expressing an antisense RNA, the strand whichwas initially the matrix strand becomes the coding strand, and viceversa. The recombination product will consequently transcribe an mRNAwhose base sequence is complementary to all or a portion of the sequenceof the mRNA for the enzyme. Consequently, the two RNA strands arecomplementary not only in their base sequences but also in theirorientation (5′ to 3′).

In a recombination product which expresses a sense RNA, the matrix andthe transcribed strands retain the orientation of the initial gene ofthe plant. The recombination products expressing sense RNA transcribe anmRNA having a base sequence which is totally or partially homologouswith the sequence of the mRNA. In the recombination products expressingthe functional enzyme, the whole coding region of the gene is linked totranscription control sequences capable of being expressed in the plant.

For example, the recombination products according to the presentinvention may be manufactured as described below. A suitable vectorcontaining the desired base sequence for the transcription, inparticular such as a DNA clone which is complementary to TOCB, istreated with restriction enzymes to cut the sequence. The DNA thusobtained is then cloned, in an inverted orientation if so desired, intoa second vector containing the desired promoter sequence and the desiredstop sequence. Among the suitable promoters, mentioned may be made ofthe promoter known as 35S of the CaMV virus as an example of a promoterconsidered as being constitutive; the promoter for the polygalacturonasegene of tomato (see Bird et al., 1998, Plant Molecular Biology,11:651-662) as an example of a promoter involved in fruit regulation; oralternatively the promoter of the gene for the small subunit of ribulosebis-phosphate carboxylase, as an example of a promoter expressed ingreen tissues. The stop sequences comprise the NOS terminator of thenopaline synthase gene.

It may be advantageous to modify the enzymatic activity of the plantduring only the growth and/or ripening of the fruit. The use of aconstitutive promoter will tend to modify the level and activity of theenzyme in all the parts of the transformed plant, while the use of apromoter which is specific for a tissue will more selectively controlthe expression of the gene and will modify the activity, for example thecoloration of the fruit. Consequently, by implementing the invention,for example in capsicums, it will be suitable to use a promoter whichwill allow the specific expression during the growth and/or ripening ofthe fruit. Finally, the sense or antisense RNA will, in this case, beproduced only in the plant organs where it is desired for there to be anaction. Among the specific promoters of the growth and/or ripening offruit which may be used, mention may be made of the polygalacturonasestimulating promoter (international patent application published underNo. WO-A-92/08798), the E8 promoter (Dieckman & Fiscer, 1998, EMBO,7:3315-3320) and the fruit-specific 2A11 promoter (Pear et al., 1989,Plant Molecular biology, 13:639-651).

A twelfth subject according to the invention relates to a plant celltransformed with a vector defined in the tenth or eleventh subjectaccording to the invention.

A person skilled in the art of plant genetic engineering is nowadaysfully aware of the various techniques for obtaining genetically modifiedplants. It is known that the plant wall constitutes a natural mechanicalbarrier that is particularly effective against the penetration of anyforeign matter into the cell and, in particular, against the penetrationof DNA. The various specific techniques for introducing DNA into plantcells are, for example, the use of the bacterium Agrobacteriumtumefaciens, the electroporation of protoplasts, the microinjection ofnaked DNA, the use of a biolistic or particle gun, or the transformationof protoplasts.

In order to be able to select the cells which have effectively beentransformed, a marker gene is introduced, in addition to the geneencoding the desired character. A gene which imparts resistance to anantibiotic will preferably be selected. In this case, the cells areselected by culturing on a medium containing this antibiotic. Only thecells containing the resistance gene may multiply.. The presence of thegene of interest may also be confirmed by hybridization with DNAcomplementary to the DNA introduced.

The recombination product according to the invention is transferred intoa target plant cell. The target plant cell may be a portion of a wholeplant or may be an isolated cell or a portion of a tissue which may beregenerated inside a whole plant. The target plant cell may be chosenfrom any species of monocotyledon or dicotyledon plant. Suitable plantscomprise any fruit-bearing plant, in particular such as tomatoes,mangoes, peaches, apples, pears, strawberries, bananas, melons,capsicums, pimentas, paprika, plants having foliage, flowers or anyother organ in which it is desired to modify the carotenoid content.

The recombination products according to the invention may be used totransform any plant, using any technique that is suitable fortransforming plants according to the invention. The cells ofmonocotyledon and dicotyledon plants may be transformed in various waysthat are known to those skilled in the art. In most cases, the cells ofthese plants, particularly when they are cells of dicotyledon plants,may be cultured to generate a whole plant which reproduces thereafter togive rise to successive generations of genetically modified plants. Anyprocess which is suitable for transforming plants may be used. Forexample, dicotyledon plants, such as tomato and melon, may betransformed using the Agrobacterium Ti plasmid. Such transformed plantsmay reproduce by crossing, or by cell or tissue culture.

A thirteenth subject according to the invention relates to a plant, orplant fragment, particularly a fruit, seed, petal or leaf, comprisingcells defined according to the twelfth subject of the invention.

The plants or plant fragments that are genetically modified according tothe invention with a vector comprising a sense sequence, in particularto increase the production of carotenoids, comprise a high level ofvitamin A precursor relative to the normal level produced by the plant.

In addition to their role in the color of the plant, carotenoids alsohave a role of protecting plants against damage which may be broughtabout by high-intensity light. As a result, plants containing a higherlevel of these carotenoids by genetic modification may be of greatinterest for regions in which cultivation is carried out with largechanges in temperature.

The genetically modified plants may have various colors, depending onwhether the carotenoid synthesis has been increased or reduced. Moreparticularly, the TOCB recombination products may be used to stimulateor inhibit the production of the colors associated with the carotenoidsproduced during the desaturation reactions, for example lycopene red, orproduct derivatives such as the yellow/orange color associated withbeta-carotene. Stimulation of the production of beta-carotenes, with anoverexpression sense recombination product, may make it possible toproduce capsicums of yellow/orange color, or alternatively a colordetermined by a beta-carotene derivative such as a more intense red, dueto the biosynthesis of capsorubin or capsanthine. The capsicums obtainedwill be found to be more appetizing by consumers.

As examples of genetically modified plants according to the presentinvention, mention will be made more particularly of fruit-bearingplants. The fruit of these plants may thus be made more appealing toconsumers by stimulating or intensifying a specific color inside. Asother plants which may be genetically modified, mention may be made oftubers such as radish, turnip and potato, and also cereals such as corn,wheat, barley and rice.

The genetically modified plants according to the invention may alsocontain other recombination products, for example recombination productshaving other effects, in particular on the ripening of fruits. Forexample, fruit having a more intense color, modified according to thepresent invention, may also contain recombination products, either whichinhibit the production of certain enzymes such as polygalacturonase andpectin esterase, or which interfere with the production of ethylene.Fruit which contain these two types of recombination products may beproduced, either by successive transformations, or by crossing twovarieties which each contain one of the recombination products, followedby selecting, from the descendents, those which contain the tworecombination products.

A fourteenth subject according to the invention relates to a process formodifying the production of carotenoids in a plant, either by increasingthe production of carotenoids, or by reducing or inhibiting theproduction of carotenoids by the plant, relative to the normal contentof carotenoids produced by the plant, said process comprising thetransformation of cells of said plants to be transformed with a vectordefined in the tenth and eleventh subject according to the invention.

A fifteenth subject according to the invention relates to a process forproducing carotenoids in a plant cell, or eukaryotic or prokaryoticcell, said process comprising the transformation of cells of saidplants, eukaryotic or prokaryotic cells to be transformed with a vectordefined in the tenth subject according to the invention.

The beta-carotenes produced by a eukaryotic or prokaryotic organismexpressing a recombination product encoding the TOCB enzyme, may beextracted in order to be used as a colorant, antioxidant or vitamin Aprecursor.

Finally, the invention also relates to a process for selecting compoundsof herbicidal nature, in which said agent is placed in contact withcells or cell membranes, in particular cells of the invention, and areduction in the consumption of oxygen by the membranes of said cells,which is associated with the inhibition of the terminal oxidaseassociated with carotenoid biosynthesis, is observed. Suitabletechniques for making this observation are illustrated in particular inExample 6.

FIG. 1 shows the cDNA sequence (SEQ ID NO: 1) and the correspondingamino acid sequence (SEQ ID NO: 2) of TOCB. The N-terminal potentialtransit peptide of the chloroplast is underscored. The probable cleavagepoint is indicated by an asterisk (*). The open triangles indicate theposition of the introns.

FIG. 2 shows the comparison between the TOCB protein (residues 111-299of SEQ ID NO: 2) and the AOX protein of soybean (SEQ ID NO: 8). (+)indicates the similar amino acids. The amino acids shown in a box formpart of the predicted transmembrane helix domains. The iron-bindingmoieties are overscored.

FIG. 3 shows the alignment of the amino acid sequences for tomato (T)(SEQ ID NO: 9), capsicum (P) (SEQ ID NO: 10) and Arabidopsis (A) (SEQ IDNO: 2) and the consensus sequence. In this consensus sequence, theconserved amino acids are indicated in upper letters and the relativelyconserved amino acids are indicated in lowercase letters.

FIG. 4 represents the oxygen consumption in isolated E. coli cellmembranes for control cells transformed with a cloning vector of theinvention and for cells expressing the product of the “IMMUTANS” gene(plastid terminal oxidase).

EXAMPLE 1 Detail of the Cloning of the Locus Encoding the TOCB Protein

1—Isolation of the Mutant

Mutation was induced by using a transposon introduced into the genome ofthe plant Arabidopsis thaliana cultivar landsberg-erecta.

This technique is largely described in an article (Long, D., Martin, M.,Sundberg, E., Swinburns, J., Puangsomlee P., and Coupland, G. (1993) Themaize transposable element system Ac/Ds as a mutagen in Arabidosis:Identification of an albino mutation induced by Ds insertion. Pro. Natl.Science USA, 10, 10370-10374) and has been used by others in thelaboratory of George Coupland at the John Innes Centre for PlantScience, Colney, Norwich, NR4 7UH, Nordwich [sic], Great Britain.

The transposition of the dissociator (Ds) transposable element used herewas triggered by producing the transposase protein (or transposase ofthe activator element, Ac).

Among the descendents of a plant which has undergone the transpositionof the element Ds, several plants having the albino mutant appearance,which differs from the wild-type plant by the absence of greenpigmentation (chlorophyll), were identified. Plants of wild-typeappearance but which transmit the mutation to their descendents werealso identified. These plants are identified as heterozygotes, bearingthe mutation on only one chromosome. The homozygous plants have a mutantphenotype and bear the mutation on the two homologous chromosomes.

2—Test of Binding of the Mutation to the Transposable Element Ds

This experiment was carried out with the aim of proving that themutation observed is caused by the insertion of the element Ds into agene which is required for correct functioning of the plant and for itswild-type appearance.

The transposable element, or transposon, Ds, is constructed so as tobear a gene for resistance to the antibiotic hygromycin (described inthe preceding references). The descendents of 35 heterozygous plantswhich bear the albino mutation were grown on an agar medium containing alethal dose of hygromycin; all the plants which bear the mutation arealso hygromycin-resistant. The conclusion is drawn therefrom that themutation is associated with the resistance gene borne by the transposon.

A portion of DNA from a plant resistant to the antibiotic hygromycin,adjacent to the transposon, was isolated. This was carried out accordingto the IPCR or inverse PCR method described in the preceding references.

By means of a “Southern blot” experiment, it was noted that the lineswhich bear the mutation have an alteration in the genomic DNA. Thisalteration is revealed when the portion of isolated DNA adjacent to thetransposon is used as a “probe”.

3—Isolation of the Gene

Using a method for screening a genomic DNA library, a clone was isolatedcontaining a genomic DNA fragment which may contain the unalteredwild-type version of the interrupted gene in the mutant.

The DNA library screened was constructed. It is described in thepublication by Whitelam, G. C., Johnson, E., Peng, J., Carol P.,Anderson, M. L., Cowl, J. S. & Harberd, N. P. (1993) Phytochrome A nullmutants of Arabidopsis display a wild-type phenotype in white light. ThePlant Cell 5, 757-768.

The total sequence of a restriction fragment obtained by enzymaticdigestion of the genomic DNA clone with the enzyme EcoR I wasdetermined. The sequence obtained covers 3000 base pairs. Among these3000 base pairs, a portion identical to the sequence of the borderfragment isolated beforehand is found, confirming the identity betweenthe isolated DNA and the gene interrupted with the transposon.

4—Isolation and Characterization of the Coding Sequence

A cDNA library was used, which is a commercial library sold by ClontechLaboratories, Inc. This is a cDNA library made from mRNAs extracted fromArabidopsis thaliana, transformed into cDNAs and then cloned into theplasmid vector pGAD10.

Using this cDNA data library, and according to the usual techniques,using the gene identified above as a probe, several clones containing acDNA of about 1400 base pairs in size were isolated.

The total sequence of the cDNA was determined and showed that this cDNAis entirely within the genomic DNA fragment identified previously. Thecoding portion (or exons) and the noncoding portion (introns) of thegene were placed on the sequence of the gene. The gene bears 9 exons and8 introns. The insertion of the transposon Ds was identified at thestart of the second exon and thus interrupts the coding portion of thegene.

The cDNA sequence has a potential start codon followed by an openreading frame of 350 amino acids, encoding a potential protein of 39 kDaknown as TOCB. A search for homology using the blastp program [(Altshulet al. (1997), Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs Nucleics Acids Res. 25, 3389-3402] revealed alow but significant homology with polypeptides belonging to the familyof mitochondrial alternative oxidase or terminal oxidase (AOX) proteins.No other significant homology was found. The homology starts at aminoacid 111 and shows 29% identity (45% similarity) with soybean oxidase.Despite the low identity with the AOX protein, a computer search forsecondary structures and potential domains of biological significancerevealed a structural similarity between the protein TOCB and AOX.Transmembrane helix domains found in AOX are located in similarpositions on the peptide sequence of TOCB, suggesting a membranelocation of TOCB and also a configuration similar to that of AOX in themembrane. Furthermore, an iron-binding moiety is conserved between TOCBand AOX. The alignment of the sequences between the proteins TOCB andAOX shows an insertion of 19 amino acids into the TOCB protein whichcorresponds to a portion of the exons 7 and 8.

The N-terminal sequence of the TOCB protein has the characteristics of achloroplast transit peptide, which is rich in leucine, arginine andserine/threonine. A computer analysis of the transit peptide potential(psort software, Nakai and Kanehisa, 1992) suggested a possible targetfor TOCB in the thylakoid compartments of the chloroplast.

5—Identification of the Mutation

The appearance of the mutant is similar to that of a mutant alreadydescribed in the literature: the “immutans” mutant, Wetzel C. M., JiangC-Z., Meehan L. J., Voytas D. L., Rodermel S. R. (1994)Nuclear-organelle interactions: the immutans variegation mutant orArabidopsis is plastid autonomous and impaired in carotenoidbiosynthesis, Plant Journal 6, 161-175.

The “immutans” mutant (spotty allele, cf. preceding reference) wascrossed with that which was isolated according to the invention. Thedescendents of the crossing is of mutant appearance, which is anexpected result if the two mutations affect the same gene. It may thusbe asserted that the gene identified corresponds to the wild-typeversion of the IMMUTANS locus and that the mutant obtained bears aninterrupted version of the gene, the product of which is thus inactive.

The first subject of the present invention thus differs from the abovemutant in that it encodes a protein whose enzymatic activity isidentical or equivalent to that of TOCB, while the product encoded by“immutans” has no activity.

EXAMPLE 2 Construction of a Vector of the Invention by Introduction ofcDNA Encoding Capsicum TOCB into a Plant Expression Vector

The vector pBI121 (sold by Clontech Laboratories, Inc.) is a vector thatis suitable for this construction.

It comprises a T-DNA region which the bacterium Agrobacteriumtumefaciens can transfer into the plant genome.

This T-DNA region comprises, inter alia, a constitutive promoter (thepromoter known as 35S from CaMV virus), the GUS gene followed by the NOSterminator (of the nopaline synthase gene). As the GUS gene is of nointerest in the invention, it is replaced with a cDNA encoding TOCB.This cDNA will thus be placed under the control of the 35S promoter andthe NOS terminator.

Any other constitutive or nonconstitutive promoter (in the latter case,it will need to be specific for the organ whose properties it is desiredto modify) and any other terminator may also be used.

A cDNA encoding TOCB was initially subcloned into the NotI restrictionsite of the bacterial plasmid pBluescriptKS: it was thus flanked by a 5′BamHI cleavage site and a 3′ SacI cleavage site.

This cDNA is excized from the plasmid pBluescriptKS with the restrictionenzymes BamHI and SacI. This BamHI-SacI fragment is inserted into thevector pBI121 which is itself cleaved with these enzymes: the BamHI siteis at the 3′ end of the 35S promoter and at the 5′ end of the GUS gene,and the SacI site is at the 3′ end of the GUS gene and at the 5′ end ofthe NOS terminator.

After ligation, the derivatives of the vector pBI121 in which the cDNAencoding TOCB (that is to say without intron) has replaced the GUS gene,are selected.

EXAMPLE 3 Transformation of a Plant Cell to Obtain a Transformed Cell ofthe Invention

The plant transformation vector derived from pBI121 obtained in Example2 is introduced into the strain of Agrobacterium LBA4404 byelectroporation. The recombinant strain is selected in the presence of50 μg/ml of kanamycin.

This transformed strain of Agrobacterium is used for the transformationof plant cells, for example tobacco cells.

The technique used to do this, which may be replaced by any othertransformation technique, is that of infecting foliar disks of tobaccoplantlets cultivated in vitro. The transformed plant cells are selectedin the presence of kanamycin. Agrobacterium is eliminated by theantibiotic cefotaxime. The foliar disks are cultivated on plant culturemedium in the presence of plant hormones (auxin and cytokinins) whichpromote the growth of cals. The cals derived from the growth of thetransformed cells are used for the regeneration of whole plants by theconventional techniques. For example, the cals are transferred ontoplant culture medium in the presence of cytokinin to induce theformation of shoots. These shoots are then cut up and transferred ontohormone-free plant culture medium in order to regenerate roots. Theantibiotics kanamycin (to select for the growth of transformed tissues)and cefotaxime (to completely eliminate Agrobacterium) are maintainedthroughout these culturing phases.

The transformed plants are placed in sterile culture in the presence ofkanamycin and cefotaxime and are then transferred to soil and cultivatedin a greenhouse until the seeds are harvested. The presence of thetransgene was confirmed by hybridization of the genomic DNA of theseplants with a specific probe derived from the transformation vectorused.

EXAMPLE 4 Cloning and Characterization of cDNA of Capsicum and TomatoFruit Corresponding to the Terminal Oxidase Associated with CarotenoidBiosynthesis (TOCB) Enzyme

The “immutans” cDNA portion of Arabidopsis encoding the mature TOCBpeptide was used as a probe to search for a cDNA library for greenpepper or red pepper under nonstringent conditions. All the positiveclones which were analyzed appeared to be derived from the same gene, assuggested by the identical sequences observed in the nontranslated 3′region. The DNA sequence of the whole clone is presented in the sequencelisting under the identifier SEQ ID NO:3. The deduced amino acidsequence is presented in the sequence listing under the identifier SEQID NO:4. The capsicum cDNA was then used to isolate the correspondingcDNA from a red tomato cDNA library (SEQ ID NO:5).

FIG. 3 shows the comparison between the abovementioned deduced aminoacid sequence and the sequences of capsicum and Arabidopsis TOCB.

The transit peptides used for targeting in the plastids revealed asequence similarity, with the exception of the N-terminal region and ofthe region close to the assumed cleavage site (ATR/Q-AT). However, themature TOCB polypeptides share a strong sequence similarity, which meansthat they have the same properties.

An alignment of the TOCB sequences also revealed the presence of twoconserved potential transmembrane domains, separated by a highlyconserved hydrophilic segment. The N-terminal domain is essentiallyhydrophilic and contains a long weakly conserved amino acid segment. TheC-terminal domain is also mainly hydrophilic and contains a conservedmoiety (EAEH) which matches a putative iron-binding site (ExxH). Inaddition, the region contains 6 cysteine residues that are conserved inTOCB, while the rest of the polypeptide lacks cysteine residues.

Some of these cysteine residues may be involved in the covalentdimerization of the protein.

EXAMPLE 5 Expression of the TOCB Genes During Ripening of the Fruit inCapsicums and Tomatoes

In order to define the mechanisms of expression of the TOCB genes, thetotal RNA was extracted from fruit at different stages of ripening. Theexpression mechanism was determined by reverse transcription of thetotal RNA, followed by a polymerase chain reaction (RT-PCR).

The TOCB gene is expressed during the growth and ripening of thecapsicum fruit. In addition, it has an expression mechanism which issimilar to that of genes encoding carotenoid desaturases, that is to sayphytocene desaturase and zeta-carotene desaturase. An increase in thelevel of transcription is observed between the unripe green stage andthe ripe green stage (fruit of an adult size), followed by anotherincrease between the ripe green stage and the degradation stage (earlyvisible signs of a color change). The level of transcription thenremains fairly constant (with a slight decrease during the reddeningstep).

The TOCB gene is also expressed during the growth and ripening of fruitin tomatoes. In tomatoes, there is also an expression mechanism which issimilar to that of the genes encoding carotenoid desaturases (phytoenedesaturase and zeta-carotene desaturase). An increase in the level oftranscription is observed between the unripe green stage and the ripegreen stage (adult-sized fruit), followed by another, greater increasebetween the ripe green stage and the degradation stage.

When the imprint of the protein of the capsicum and tomato fruit wasdesired, using antibodies directed against TOCB, this polypeptide wasfound at various stages of development of the fruit. These testsdemonstrated an increase in the level of the TOCB protein, from the ripegreen stage to the degradation stage. This level of protein remainedhigh throughout the ripening of the fruit.

These results demonstrate that the TOCB genes are expressed and that theTOCB protein is present in the fruit. In a manner similar to that of thestructural enzymes involved in the desaturation of are accumulatedduring the ripening when the carotenoid biosynthesis is increased.

The results presented in the description reveal that TOCB is an elementof the carotenoid biosynthesis system.

It may be envisaged to use the TOCB protein to modify carotenoidbiosynthesis, in particular in plant tissues or cells or in bacteriawhich have an inefficient or poorly efficient carotenoid biosynthesissystem. TOCB may be produced at the same time as the structural enzymesof carotenoid biosynthesis to increase the efficacy of the production ofcarotenoids.

EXAMPLE 6 Catalytic Properties of TOCB Analyzed After its Expression inE. coli

A synthetic product consisting of the region encoding the mature TOCBpolypeptide from Arabidopsis was inserted into a prokaryotic expressionvector (such as pQE31, sold by QIAGEN, it being understood that anyother vector would give identical results).

The coding region intended to be inserted into the expression vector maybe obtained by cleavage using restriction enzymes which act close to thecodons corresponding to the site of cleavage of the transit peptide.

Alternatively, an amplification by PCR of the coding region may becarried out. The following oligonucleotides will advantageously be usedto amplify the sequence of Arabidopsis TOCB:

5′-GCAACGATTTTGCAAGACG-3′ (SEQ ID NO: 6) and

5′-TTAACTTGTAATGGATTTCTTGAG-3′ (SEQ ID NO: 7).

Other assembly products comprising the region encoding TOCB in otherspecies (such as capsicum or tomato) may also be used.

These plasmids may be introduced into E. coli cells according toconventional techniques. In order to obtain the recombinant protein inE. coli, the cells are cultured under the following conditions: 10 ml ofan overnight preculture in a rich medium are deposited in 300 ml of M9medium (Na₂HPO₄ 34 mM, KH₂PO₄.22 mM, NH₄Cl 18 mM, NaCl 8.5 mM, MgSO₄ 1mM, CaCl₂ 0.1 mM, thiamine 1 mM) containing 0.2% of glycerol and thesupply of antibiotic required to stop the growth of the cells which havelost the plasmid. The growth of the bacteria is continued at 37° C. withvigorous agitation up to the half-exponential growth phase, preferablyuntil an optical density of 0.3 at 600 nm is read.

After inducing this chimeric gene with the inducer IPTG and adding 1mg/l of FeSO₄, the culture is maintained at 25° C. with vigorousagitation for 3 hours. The cells are then harvested by centrifugation at4° C., washed with 10 mM MgCl₂, 0.75M sucrose, 20 mM Tris-HCl, at pH7.5, and centrifuged again. The cells are then suspended in 0.75Msucrose, 20 mM Tris-HCl, at pH 7.5, and lysed by addition of lysozyme(0.2 mg/ml) and EDTA (25 mM) at 30° C. for 30 minutes, and thensubjected to an osmotic shock by addition of two volumes of water, afterwhich they are treated with ultrasound at 0° C. A standardcentrifugation in a centrifuge at slow speed makes it possible to removethe nonlysed cells and the debris. A high-speed centrifugation (forexample in a Beckman 50 Ti rotor at 40000 rpm) at 4° C. produces amembrane which is suspended in 0.75M sucrose, 20 mM Tris-HCl, at pH 7.5,and maintained at 4° C.

To test the enzymatic activity of the TOCB, the consumption of oxygen bythe resulting membranes is measured using a standard oxygen electrodeand is expressed in nmol of O₂ consumed per minute and per gram ofprotein.

As shown in FIG. 4, the addition of NADH induces the consumption ofoxygen both in the control membrane (transformed with the cloningvector) and in the membrane containing the TOCB. This oxygen consumptionincreases when 0.2 mM plastoquinone is added. The addition of KCNgreatly inhibits the oxygen consumption in the control membranes. In themembranes containing TOCB, a high cyanide-resistant oxygen consumptionis observed. This reflects the plastoquinol:oxygen oxidoreductaseactivity of the TOCB, which activity may be inhibited by adding 0.5 mMn-propyl gallate (nPG). The addition of nPG (0.5 mM) to the controlmembrane before KCN does not produce an effect, indicating that thecompound does not interfere with the normal flow of electrons in the E.coli membranes (FIG. 4).

This test may be used to study the inhibitory power of a compound onTOCB activity. Thus, an inhibitor may be controlled when it has noeffect on the endogenous respiratory chain of E. coli, in particular onthe complex I of the chain which oxidizes NADH. Nevertheless, if such isthe case, NADH may be replaced with succinate as an electron donorwithout passing via the complex I. Any inhibitor of TOCB activity may betested on suitable plants, by watering the soil, adding a culture mediumand applying directly to the leaves, with respect to the inhibition ofcarotenoid biosynthesis, resulting in bleaching, and may thus find anapplication as a herbicide.

The test described may be modified to carry out a large-scale screeningof inhibitors of TOCB activity, and their application as herbicides. Inthis case, measurement of the oxygen consumption using an oxygenelectrode will preferably be replaced with another method ofmeasurement.

The oxidase activity of TOCB may be determined by measuring theconsumption of NADH during the reaction, for example byspectrophotometry, by measuring the absorbance at 340 nm. Theconsumption of NADH and the production of NAD during the test shouldresult in a decrease in the absorbance at 340 nm. Alternatively, anyspecific coloration of NAD or of NADH may be used to monitor changes inNAD or NADH during the test.

If succinate is used as an electron donor in the test, the respiratoryactivity of the bacterial membranes will result in the oxidation of thesuccinate to fumarate. In this case, the activity of the TOCB may bemonitored in the presence of KCN, by measuring the concentrations ofsuccinate and fumarate which change during the test.

According to another possibility, an artificial electron donor may beused. An example of this is phenazine metasulfate (PMS). It may beoxidized by the succinate dehydrogenase of the bacterial membranes; itis colorless in the reduced form and yellow in the oxidized form.

Samples of bacterial membrane containing TOCB oxidize PMS in thepresence of KCN. An inhibitor of TOCB activity will prevent theappearance of the yellow color due to the oxidation of the PMS. Thistest, which is simple to perform, may be carried out in multi-wellplates, allowing a bulk screening of molecules capable of inhibiting theactivity of TOCB to be performed.

1. A process for modifying the production of carotenoids in a plant, by increasing the production of carotenoids relative to the normal content of carotenoids produced by the plant, said process comprising transformation of cells of said plant with a vector comprising: (1) a nucleotide sequence encoding an enzyme having terminal oxidase activity involved in carotenoid biosynthesis, said enzyme comprising SEQ ID NO: 2, or (2) a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1 and encoding an enzyme having terminal oxidase activity involved in carotenoid biosynthesis, wherein said nucleotide sequence is operably linked to a promoter, such that the vector can generate mRNA in the plant cells, and the production of carotenoids in said plant is increased.
 2. A process for producing carotenoids in a plant cell, or eukaryotic or prokaryotic cell, said process comprising transformation of at least one plant, eukaryotic or prokaryotic cell with a vector comprising: (1) a nucleotide sequence encoding an enzyme having terminal oxidase activity involved in carotenoid biosynthesis, said enzyme comprising SEQ ID NO: 2, or (2) a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 1 and encoding an enzyme having terminal oxidase activity involved in carotenoid biosynthesis, wherein said nucleotide sequence is operably linked to a promoter, such that the vector can generate mRNA in said at least one cell, and the production of carotenoids in said plant, eukaryotic or prokaryotic cell is increased.
 3. The process according to claim 1, wherein said vector comprises a nucleotide sequence encoding SEQ ID NO:
 2. 4. The process according to claim 2, wherein said vector comprises a nucleotide sequence encoding SEQ ID NO:
 2. 5. The process according to claim 1, wherein said vector comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 1 and encoding an enzyme having terminal oxidase activity involved in carotenoid biosynthesis.
 6. The process according to claim 2, wherein said vector comprises a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 1 and encoding an enzyme having terminal oxidase activity involved in carotenoid biosynthesis. 